Eukaryotic translation initiation factor 2 (elF-2) catalyzes the first step in the initiation of protein synthesis through the formation of a ternary complex between the 40S subunit of the ribosome, the initiator tRNA, and GTP. Regulation of the activity of eIF-2 is a common final step in a number of pathways that regulate the overall rate of translation with the metabolic needs of the cell. Increased phosphorylation of eIF-2 alpha and translational repression have been described in viral infection, growth and differentiation, and metabolic alterations. To examine the regulation of eIF-2 expression at the transcriptional level, we have cloned and characterized the promoter for the gene for the alpha subunit of eIF-2. We are interested in determining which transcription factors interact with this gene and RNA polymerase Ii. Analysis of the promoter using a variety of techniques has revealed evidence for a number of specific protein-DNA interactions. We have identified a cluster of five DNase I hypersensitive sites within a 25 kb region surrounding the promoter. These sites are found in all cells types examined. Associated with these sites are a number of cis elements identified by in vitro DNase I footprint experiments which interact with proteins present in nuclear extracts of K562 cells and TIL cells. None of these elements shares sequence homology with the binding sites of known regulatory factors, suggesting that eIF-2 alpha transcription may be regulated through a novel series of regulatory elements and factors. The most prominent element consists of two adjacent protein binding sites composed of palindromic sequences, Mutation of this sites reduces expression of eIF-2 alpha. The protein binding to this element, termed the alpha palindrome binding protein or alpha-PAL, has been purified to homogeneity from K562 and TIL nuclear extracts using conventional and DNA affinity chromatography. This study may ultimately have implications for human disease therapy. Successful gene therapy requires proper regulation of the transferred gene, and the work described in this study is directly aimed at understanding transcriptional regulation. In addition, eIF-2 alpha is an essential housekeeping gene; as a group, housekeeping genes have been relatively little studied. A detailed understanding of transcriptional regulation may also facilitate the identification of therapeutic agents which act through alterations in specific gene expression.